Purification and characterization of a streptomycete collagenase

A soil streptomycete designated as Streptomyces sp. A₈ produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase'as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as α-α-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetracetate completely inhibited the enzyme activity. Among the cations tested only Ca^{2 +} and Mg^{2 +} enhanced the collagenase activity. Heavy metal ions like Pb^{2 +}, Ag⁺, Cu^{2 +} and Zn^{2 +} strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca^{2 +}. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Characterization of soluble rifamycin oxidase from Curvularia lunata var. aeria

Curvularia lunata var. aeria was grown on yeast extract, peptone and carboxymethylcellulose (YPC) medium for the production of extracellular rifamycin oxidase. The enzyme was partially purified through a Sephadex G-75 column. The half lives of rifamycin oxidase at 30° and 40°C were 9 d and 100 min, respectively. The activation and deactivation energies of the partially purified enzyme, calculated from Arrhenius plots, were 5.80 and 35.10 kcal mol^-1 respectively. The enzyme exhibited a K _m (rifamycin B) value of 0.67 mmol l^-1 and a V _max of 11 μmol h^-1 ml. Three metal ions, Fe²⁺, Ag⁺ and Hg²⁺, inhibited the enzyme in the 10–20 mmol l^-1 metal ion concentration range. Catalytic activity was not affected by the chelating agent, EDTA.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Interaction between monobactams and model d-alanyl-d-alanine-cleaving peptidases

Abstract Several monobactams reacted with the serine dd -peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the Michaelis complexes formed between the R61 enzyme and sulfazecin (32 μM) and between the R39 peptidase and SQ 26324 (0.35 μM) had the lowest values ever observed with any β-lactam compound, suggesting an excellent fit of these two monobactams with the active sites of the respective enzymes. Azthreonam had a very poor inactivating potency, confirming its high selective reactivity towards the penicillin binding protein No. 3 of Escherichia coli . The Zn²⁺ dd -peptidase (from Streptomyces albus G) had a high intrinsic resistance to β-lactam compounds whether they possessed a mono- or a bicyclic structure.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber ( Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg²⁺. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg²⁺. The NR activation state, i.e. activity in the presence of Mg²⁺ relative to activity in the absence of Mg²⁺, increased with light intensity up to 400 μmol m⁻² s⁻¹ PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg²⁺. The ATP-inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5'-AMP. These results support the view that inactivation/activation of cucumber-leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5'-AMP was also observed when activity was assayed in the absence of Mg²⁺, thus indicating that 5'-AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg²⁺ and in the −Mg²⁺ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.     

An extracellular α-L-arabinofuranosidase secreted from cell suspension cultures of carrot

Carrot ( Daucus carota L. cv. Kintoki) cell cultures secrete an α-L-arabinofuranosidase (α-L-AFase, EC 3.2.1.55) into their culture medium during growth. The extracellular α-L-AFase (α-L-AFase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200HR and Concanavalin A-Sepharose, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 84 kDa by Sephacryl S-200HR gel-permeation, and 80 kDa by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed for amino acid composition and the sequence of the first 21 amino acids of the N-terminus. The isoelectric point was pH 5.6, the pH optimum 3.8, and the temperature optimum 55°C. The activity was inhibited by Zn²⁺, Ag²⁺, Cu²⁺, Hg²⁺ and p -chloromercuribenzoate. The K_m and V_max values for p -nitrophenyl-α-L-arabinofuranoside were 0.22 m M and 0.11 mmol (mg protein)⁻¹ h⁻¹, respectively. The enzyme acted on beet arabinan in an exo-fashion, and was capable of hydrolysing arabinose-rich polymers purified from pectic polysaccha-rides of carrot cell cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.     

Protein kinase activities in ripening mango (Mangifera indica) fruit tissue. II. Purification and characterization of a casein kinase I

A protein kinase (PK‐II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange‐, dye‐ligand, and gel filtration chromatography. The enzyme was purified over 500‐fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 µmol mg⁻¹ protein min⁻¹ with ATP as phosphoryl donor. SDS‐PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn²⁺ could not substitute for the Mg²⁺ needed for activity and Ca²⁺ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI‐7, ( N ‐[2‐aminoethyl]‐5‐chloroisoquinoline‐8‐sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a K_m (ATP) and K_m (casein) of 14 µ M and 0.18 mg ml⁻¹, respectively, with a K_i (ADP) of 3.2 µM. The enzyme can be classified as a casein kinase I type of protein kinase (EC 2.7.10).     

Involvement of Na+,K+-ATPase in the Mitogenic Effect of Insulin-Like Growth Factor-I on Cultured Rat Astrocytes

Abstract: We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the α1 isoform of Na⁺,K⁺-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na⁺,K⁺-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na⁺,K⁺-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [³H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na⁺,K⁺-ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. ¹²⁵I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca²⁺-free medium. The stimulation by IGF-I of [³H]thymidine incorporation was attenuated by ouabain and a low external K⁺ level. These findings suggest that stimulation of Na⁺,K⁺-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.     

Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

α-Galactosidase from the yeast Torulaspora delbrueckii IFO 1255

The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulaspora species. The strain IFO 1255 produced extracellular and cell-associated forms of α-galactosidase when grown on either melibiose or galactose as the sole carbon source. Most of the enzyme was located outside of the cell membrane: the periplasmic space, or cell walls, or both. α-Galactosidase was purified to homogeneity from the cell-free extract of the strain IFO 1255 by acid treatment and column chromatography on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M. The molecular weight of the purified enzyme was estimated to be 88 000 by SDS-polyacrylamide gel electrophoresis and 530 000 by gel filtration. The enzyme contained 50% of its molecular weight as carbohydrate. Optimum pH and temperature were 4.5–5.5 and 55°C, respectively. The enzyme was inhibited strongly by Ag²⁺, Hg²⁺ and Cu²⁺ each at 1 mmol 1^-1. The K _m (μmol 1^-1) for p -, o -, m -nitrophenyl α-D-galactopyranoside, melibiose, raffinose and stachyose were 2.8, 1.3, 2.8, 4.2, 170 and 230, respectively, and V _max (μmol min^-1 mg protein^-1) for those substrates were 310, 140, 21, 22, 30 and 44, respectively. The properties of α-galactosidase from T. delbrueckii IFO 1255 were similar to those from the related species, Saccharomyces cerevisiae.     

Properties of NADP+-malic enzyme from pod walls of chickpea (Cicer arietinum)

NADP⁺-malic enzyme ( l -malate: NADP⁺ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn²⁺ or Mg²⁺, for its activity. K_m values at pH 7.8 for malate, NADP⁺ and Mn²⁺ were 4.0, 0.031 and 0.71 m M , respectively. Mn²⁺-dependent activity was inhibited by heavy metal ions such as Cd²⁺, Zn²⁺, Hg²⁺, and to a lesser extent by Pb²⁺ and Al³⁺. Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP⁺ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP⁺, pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP⁺-malic enzyme is controlled by intracellular concentrations of substrates and products.